PCM, the L-isoaspartyl protein carboxyl methyltransferase, is a protein-repair enzyme that repairs damage caused by spontaneous formation of isoaspartate (isoAsp) from aspartate or asparagine. PCM contributes to longevity in a variety of organisms, and defects in isoAsp repair have been linked to important pathological conditions, including autoimmunity, Alzheimer'sdisease, epilepsy, spina bifida and cancer. Although it is clear that PCM repairs proteins and restores activity in vitro, important questions about the enzyme's roles in vivo remain to be answered. The proposed research, to be conducted by the principal investigator and 12-15 undergraduate students over the next three years, will address how, where and when PCM acts to protect stationary-phase Escherichia coli bacteria (a model system for aging) from the deleterious effects of isoAsp damage. Two specific questions will be addressed: Aim 1: How does PCM enhance survival of aging E. coli? The hypothesis that isoAsp damage acts synergistically with oxidative or other stresses to destabilize protein conformation will be tested by: (i) a genetic approach to identify interactions between PCM-mediated protein repair and other maintenance systems; (ii) in vivo measurement of protein aggregation and oxidation; and (iii) an in vivo protein folding reporter used to probe conformation of potential PCM substrates. Aim 2: When and where does PCM exert its effect? The hypothesis that PCM is advantageous primarily after restoration of nutrients allows resumption of metabolic activity and in transiently active subpopulations will be tested by (i) measuring isoAsp damage, PCM activity and related parameters in aging and recovering cells, viable and inviable subpopulations and different cellular compartments; and (ii) separating aging from recovery using a toxin-antitoxin system to induce stasis without depleting nutrients. Insights gained from this work will contribute to understanding and eventually ameliorating the effects of aging and a variety of degenerative diseases. PUBLIC HEALTH RELEVANCE: Protein damage resulting from abnormal isoaspartyl amino acids (isoAsp) can be repaired by the L-isoaspartyl protein carboxyl methyltransferase (PCM). Recent research links unrepaired isoAsp with reduced longevity, autoimmunity (e.g., lupus and diabetes), epileptic seizures and progression of brain tumors. The proposed research will contribute to understanding the mechanisms by which PCM protects cells from the effects of isoAsp damage, providing the foundational knowledge needed to better understand and eventually better treat the causes and consequences of aging and degenerative disease. [unreadable] [unreadable] [unreadable]